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J. Biol. Chem., Vol. 280, Issue 42, 35172-35183, October 21, 2005
The Translational Efficiencies of the Two Leishmania infantum HSP70 mRNAs, Differing in Their 3'-Untranslated Regions, Are Affected by Shifts in the Temperature of Growth through Different Mechanisms*From the Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain
Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 °C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 °C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 °C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 °C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 °C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 °C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the
Received for publication, May 20, 2005 , and in revised form, August 3, 2005. * This work was supported by the Spanish Ministerio de Ciencia y Tecnología (Grant BMC2002-04107-C02-01) and by an institutional grant from Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 34-914-978-454; Fax: 34-914-974-799; E-mail: jmrequena{at}cbm.uam.es.
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